EP4022039B1 - Aktivierte mesenchymale stammzellen zur verwendung in der behandlung von gliedmassenischämie - Google Patents

Aktivierte mesenchymale stammzellen zur verwendung in der behandlung von gliedmassenischämie Download PDF

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Publication number
EP4022039B1
EP4022039B1 EP19791361.9A EP19791361A EP4022039B1 EP 4022039 B1 EP4022039 B1 EP 4022039B1 EP 19791361 A EP19791361 A EP 19791361A EP 4022039 B1 EP4022039 B1 EP 4022039B1
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treated
admscs
cells
metamizole
mscs
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French (fr)
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EP4022039A1 (de
EP4022039C0 (de
Inventor
Andrus LOOG
Jekaterina Kazantseva
Olavi VASAR
Tiit MEREN
Triin VASAR
Mart RAIK
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Cellin Technologies Oue
Taastava Kirurgia Kliinik AS
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Cellin Technologies Oue
Taastava Kirurgia Kliinik AS
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Priority to HUE19791361A priority Critical patent/HUE066319T2/hu
Priority to HRP20240538TT priority patent/HRP20240538T1/hr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • Isolated stem cells can be manipulated at different stages of cell therapy procedures including cell isolation, cell propagation, conditioning for transplantation and post grafting.
  • the present invention relates to the use of clinically approved anti-inflammatory drugs to modulate activity of mesenchymal stem cells isolated from the adipose tissue. Modulation of secretion of different growth factors including angiogenic factors and cytokines as well as modulation of metabolic activity can be used to develop efficient MSC based pharmaceutical products for treatment of variety of health conditions.
  • MSCs Mesenchymal stem cells
  • Augello & De Bari, 2010 Unlike pharmaceutical treatments that deliver particular active component at a specific dose, MSCs exert therapeutic effects by secreting various bioactive compounds in response to external stimulation (Ma et al., 2014).
  • the soluble factors produced by MSCs are involved in anti-inflammatory and neovascularisation processes with profound effects on tissue injury and regeneration.
  • MSCs diseases that can be treated using MSCs are for example immune and non-immune disorders such as myocardial infarction, diabetes, graft versus host disease, and liver cirrhosis (Wei et al., 2013) (Shi et al., 2010).
  • immune and non-immune disorders such as myocardial infarction, diabetes, graft versus host disease, and liver cirrhosis (Wei et al., 2013) (Shi et al., 2010).
  • Understanding the immunomodulatory properties of MSCs and ways how inflammatory microenvironment affects their function is of immense importance for developing better strategies to increase therapeutic efficiency of MSCs with a goal to create local and/or systemic conditions to stimulate healing and tissue regeneration.
  • Peripheral arterial disease is a condition characterized by restricted supply of oxygen and glucose due to malfunction of blood vessels.
  • Peripheral vascular disease commonly affects arteries and in most advanced stages causes critical limb ischemia (CLI).
  • CLI critical limb ischemia
  • Non-steroidal anti-inflammatory drugs and synthetic glucocorticoid injections are widely used to reduce pain and inflammation of CLI patients.
  • the result of the disease progression is gangrene and limb amputation. Development of new advanced therapies could improve clinical outcome and increase the life standard of patients.
  • MSCs for the treatment of ischemic conditions have been described in several animal models and early-phase human clinical trials (Liew & O'Brien, 2012).
  • Therapeutic efficiency of MSCs depends on their ability to provide immunomodulatory and angiogenic factors to suppress inflammation and stimulate angiogenesis.
  • Non-activated MSCs express low levels of immunosuppressive factors, but the local conditions at the site of injections affect their functionality.
  • different strategies have been developed. The stimulation of MSCs by IFN ⁇ or TNF ⁇ has been used to induce the secretion of immunomodulatory factors (Crop et al., 2010).
  • Prostaglandin PGE2 is known to be one of the important compounds secreted by MSCs that is responsible for modulation of inflammation. However, the effect of NSAIDs on complex of metabolic responses and secretion of anti-inflammatory and angiogenic factors in MSC anti-inflammatory therapy is not known.
  • NSAIDs paracetamol, metamizole (analgin), ketoprofen and diclofenac
  • glucocorticoid prednisolone used in clinical practice to treat ischemic disorders, at therapeutic doses on cell cycle, metabolic activity, as well as on expression of angiogenic and inflammatory cytokines by AdMSCs has been analysed and will be described hereinafter.
  • the invention is set out in the appended set of claims.
  • the object of this invention is to provide a pharmaceutical product for angiogenesis growth to replace occluded blood vessels for avoiding limb amputation.
  • the inventors have developed protocols to stimulate mesenchymal stem cells to secret regulators that affect regeneration in ischemic limb and improve neo-vasculogenesis and arteriogenesis.
  • Treatment of MSCs with metamizole analgin
  • changes cell cycle stimulates synthesis of angiogenic trophic factors VEGF, HGF, TEK and bFGF, reduces expression of inflammatory cytokines and chemokines such as IL6, IL1RN, CCL2, IL8/CXCL8.
  • Using rat models of limb ischemia shows that treatment of MSCs by metamizole (analgin) prior to transplantation stimulates neo-vasculogenesis and arteriogenesis of the operated limb.
  • AdMSCs are perspective and promising cell source for cellular therapy to treat critical limb ischemia (CLI).
  • CLI critical limb ischemia
  • AdMSCs The priming of AdMSCs by metamizole affects the proliferation and metabolic activity of AdMSCs, changes cell cycles, dynamically modulates expression profile of inflammatory cytokines and chemokines and induces expression of angiogenic markers, important in the context of the treatment of the disease.
  • pre-clinical experiments on rats demonstrated that activated by metamizole (analgin) AdMSCs were more effective for their therapeutic applications to treat limb ischemia by their accelerated and reliable neoarteriogenesis and neoangiogenesis.
  • secretion of angiogenic growth factors VEGFA, HGF, bFGF, TEK are stimulated and the levels of pro-inflammatory cytokines IL6, CXCL8, CCL2, IL1-RN are reduced by activation of mesenchymal stem cells (MSCs).
  • MSCs mesenchymal stem cells
  • the invention provides metamizole treated mesenchymal stem cells for treating limb ischemia of a human patient.
  • the pharmaceutical product of this invention comprises metamizole treated mesenchymal stem cells.
  • the product is in injectable form and comprises 1 million cells per kg of a patient.
  • the cells are administered in an amount of 0,75 - 1,5 million per kg of body weight of a patient.
  • Metamizole treated cells are in micro concentrations.
  • a method of producing the product comprising metamizole treated mesenchymal stem cells comprises following steps:
  • MSCs produce proteins and signalling molecules for new blood vessel growth that accelerate the growth of new arteries.
  • the effects of different drugs on MSCs and the drug, metamizole, used in present invention has been studied and will be described hereinafter.
  • the product of this invention has been successfully tested on animals.
  • Stem cells are already known to be involved in neoangiogenesis and neoarteriogenesis, and the aim of previous laboratory studies and animal experiments was to find the most effective cellular drug combination. As time is crucial for patients with critical leg ischemia, it is important to find a cellular drug that works as quickly as possible. Although the product of this invention is so far tested only on animals, according to prior art, it is believed to have the same effect on human patients.
  • AIDs affect metabolic activity and cell cycle progression of MSCs
  • Fig. 2 Effect of drugs on cell cycle changes measured by flow cytometry is shown in Fig. 2 .
  • the percentage of cells in G0/G1 phase was not significantly affected upon treatment with diclofenac (81%), ketoprofen (80%) and prednisolone (77%) ( Fig. 2 ).
  • the percentage of cells in G0/1 phase was lower in the cultures treated with metamizole (analgin) (65%) and paracetamol (71%), whereas the percentage of cells in G2/M cell cycle phase was increased in these cultures ( Fig. 2 ).
  • metamizole (analgin) and paracetamol are the only studied drugs that affect cell cycle, since they accumulated the AdMSCs in the G2/M phase.
  • AIDs affect expression of angiogenic factors
  • MSCs Numerous soluble factors produced by MSCs are involved in the regulation of angiogenesis and neovascularisation in vivo (Estrada et al., 2009). On the other hand, MSCs have been revealed to inhibit angiogenesis in certain conditions (Otsu et al., 2009). Also, trophic factors VEGF and bFGF have been shown to stimulate angiogenesis in ischemia treatments (Leung et al., 1989). The effect of the AIDs on expression of angiogenic factors in AdMSCs was analyzed. Different AIDs affected the expression of angiogenic factors differently either stimulating or suppressing their expression.
  • a number of inflammatory cytokines and chemokines secreted by MSCs are involved in the process of immunoregulation, thereby affecting immunocompetent cells. Quantitative differences in the levels of cytokines secreted by MSCs determine the local conditions of the microenvironment and induce anti-inflammatory reaction. Identification of inflammatory biomarkers profiles in response to MSCs therapy coupled with AIDs treatment could predict the consequences of such intervention for immunologic status in whole. The effect of AIDs treatments on inflammatory profile of AdMSCs at the protein and gene expression levels was analyzed.
  • AdMSCs were exposed to lipopolysaccharide (LPS) prior to AIDs treatments.
  • LPS lipopolysaccharide
  • the levels of inflammatory cytokines IL6, CXCL8, CCL2 and IL1RN were increased following LPS treatment (Table 1).
  • LPS-treated AdMSCs were exposed to AIDs to study their effect on cytokines in inflammatory conditions.
  • the aim of the preclinical study was to assess conditions and cells that best promote neoangiogenesis and neoarteriogenesis by comparing differently isolated and conditionally manipulated human AdMSCs.
  • These types of preclinical studies imply the use of a hind limb ischemia model (HLIM), where the restoration of the revascularization of ischemic muscle occurs due to the regenerative potential of administrated drugs (cells) but not by animal's own regeneration capability.
  • HLIM hind limb ischemia model
  • This window-of-ischemia should last at least for 2-3 weeks.
  • Hellingman model of HLIM that is well-suited for testing of AdMSCs for in vivo regeneration was successfully developed.
  • BEAULI Lipoaspirate obtained with use of body-jet (human med, routine clinical practice for treating soft tissue defects). Lipoaspirate is obtained with use of water - jet-assisted lipoaspiration. The harvested fat is gently separated from the remaining fluid with LipoCollector ® or FillerCollector ® and has been used immediately for fat transfer. This technique has been used as clinical standard for fat cells transfer.
  • Cytori Cellution 800/CRS System Cytori Therapeutics INC.
  • Cytori Cellution-derived regenerative cells are as gold standard in regenerative medicine for enriching fat graft as well for improving angiogenesis in grafted areas.
  • Cytori Cellution 800/CRS System uses lipoaspirate, digests with collagenase, washes and separates regenerative cells with centrifugation. Before injection the amount on nucleated cells was analyzed with cell counter. Average dosage of living nucleated cells were counted using Nucleocounter NC100 (Chemometec). Average count of living nucleated cells were 0,9 ⁇ 10 6 cells/ml.
  • MSC Human AdMSCs were obtained from freshly isolated subcutaneous adipose tissue and characterized as previously reported (Lin et al., 2007). For each administration, a pool of AdMSCs from at least 3 individuals and passage number until 3 was grown in low glucose Dulbecco's modified Eagle's medium (DMEM-LG) (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (PAA, Pasching, Austria) and 1% penicillin-streptomycin (PEST) (Life Technologies). Achieving about the 80-90% confluence, cells were collected and 2 ⁇ 10 5 cells were used per animal.
  • DMEM-LG low glucose Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • PEST penicillin-streptomycin
  • MSCD AdMSCs were grown in standard culture conditions as described before until the 80-90% confluence, the medium was changed for DMEM-LG containing 1% FBS and 1% PEST at 12 h before the treatment, stimulated with LPS (0.1 ug/ml; Sigma-Aldrich, Steinheim, Germany) for 2 hours, intensively washed with phosphate buffered saline (PBS), and treated with metamizole (analgin) (10 ⁇ M) for 24 hours. Cells were collected and 2 ⁇ 10 5 cells were used per animal.
  • LPS 0.1 ug/ml
  • PBS phosphate buffered saline
  • metamizole analgin
  • SALINE In control series rats were treated with saline (0,9% NaCl) injection.
  • mice Female Sprague-Dawley (SD) rats were housed under standard animal facility conditions (2-4 animals per cage in temperature (22 ⁇ 2 ° C) and humidity (55 ⁇ 10%) controlled room with 12h:12h light:dark cycle). Animals were given ad libitum access to standard maintenance rodent diet and water.
  • Angiography was performed on the 14th day after the surgery. Animals were under the general anesthesia, on supine position, on heated and temperature-controlled surface. Midline incision was performed in abdominal wall, where abdominal aorta was exposed. MicroSlide kit (Galt Medical Corp) together with contrast medium Omnipaque 300 were used for aortic cannulation. Digital Subtraction Angiography was performed with Ziehm Vision RFD, 20 kW (Ziehm Imaging GmbH).
  • the angiography results were analyzed by two vascular surgeons who did not know the study groups. In analysis, the total amount of detectable vessels was counted, as well as the count of curled vessels to see neoarteriogenesis separately from vasculogenesis.
  • FIG. 10 Analysis of vasculogenesis between different cell treatment groups is shown in Fig. 10 .

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Claims (5)

  1. Produkt, das mit Metamizol behandelte mesenchymale Stammzellen zur Verwendung bei der Behandlung der menschlichen Gliedmaßenischämie umfasst.
  2. Produkt zur Verwendung nach Anspruch 1, wobei das Produkt in einer Dosis von 1 Million Zellen pro kg eines Patienten injiziert wird.
  3. Erzeugnis zur Verwendung nach Anspruch 1 oder 2, wobei die Zellen in einer Menge von 0,75 bis 1,5 Millionen pro kg Körpergewicht eines Patienten verabreicht werden.
  4. Erzeugnis zur Verwendung nach Anspruch 2 oder 3, wobei mit Metamizol behandelte Zellen in Mikrokonzentrationen vorliegen.
  5. In-vitro-Verwendung eines Metamizols zur Aktivierung von mesenchymalen Stammzellen (MSCs) zur Stimulierung der Sekretion der angiogenen Wachstumsfaktoren VEGFA, HGF, bFGF, TEK und zur Verringerung der Spiegel der pro-inflammatorischen Zytokine IL6, CXCL8, CCL2, IL1-RN.
EP19791361.9A 2019-08-27 2019-08-27 Aktivierte mesenchymale stammzellen zur verwendung in der behandlung von gliedmassenischämie Active EP4022039B1 (de)

Priority Applications (2)

Application Number Priority Date Filing Date Title
HUE19791361A HUE066319T2 (hu) 2019-08-27 2019-08-27 Aktivált mezenchimális õssejtek végtag-iszkémia kezelésében történõ alkalmazásra
HRP20240538TT HRP20240538T1 (hr) 2019-08-27 2019-08-27 Aktivirane mezenhimalne matične stanice za uporabu u liječenju ishemije udova

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PCT/IB2019/057182 WO2021038275A1 (en) 2019-08-27 2019-08-27 Activated mesenchymal stem cells for treating limb ischemia

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EP4022039A1 EP4022039A1 (de) 2022-07-06
EP4022039B1 true EP4022039B1 (de) 2024-04-03
EP4022039C0 EP4022039C0 (de) 2024-04-03

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US (1) US20220347223A1 (de)
EP (1) EP4022039B1 (de)
JP (1) JP7442624B2 (de)
KR (1) KR20220047874A (de)
CN (1) CN114269901A (de)
AU (1) AU2019463156B2 (de)
CA (1) CA3152963A1 (de)
ES (1) ES2982323T3 (de)
HR (1) HRP20240538T1 (de)
HU (1) HUE066319T2 (de)
PL (1) PL4022039T3 (de)
WO (1) WO2021038275A1 (de)

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TW201039815A (en) * 2009-04-13 2010-11-16 Resolvyx Pharmaceuticals Inc Compositions and methods for the treatment of inflammation
DK3366306T3 (en) * 2016-12-23 2022-01-10 Univ Istanbul Rektoerluegue Use of some peptides in diagnosis and treatment of diabetes, obesity and metabolic diseases associated thereto
WO2018236131A1 (ko) * 2017-06-20 2018-12-27 주식회사 툴젠 안지오포이에틴-1 또는 vegf를 분비하는 줄기세포 및 이를 포함하는 심혈관 질환의 예방 또는 치료용 약학 조성물
WO2019146131A1 (ja) * 2018-01-24 2019-08-01 学校法人順天堂大学 間葉系幹細胞による処置の効果を増幅するための組成物

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WO2021038275A1 (en) 2021-03-04
AU2019463156A1 (en) 2022-03-03
AU2019463156B2 (en) 2024-08-15
CA3152963A1 (en) 2021-03-04
ES2982323T3 (es) 2024-10-15
HRP20240538T1 (hr) 2024-07-05
PL4022039T3 (pl) 2024-08-12
CN114269901A (zh) 2022-04-01
US20220347223A1 (en) 2022-11-03
KR20220047874A (ko) 2022-04-19
EP4022039A1 (de) 2022-07-06
EP4022039C0 (de) 2024-04-03
JP7442624B2 (ja) 2024-03-04
JP2022548500A (ja) 2022-11-21
HUE066319T2 (hu) 2024-07-28

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